Selective extraction of phospholipids from soybeans with supercritical carbon dioxide and ethanol

Abstract

Supercritical carbon dioxide (SC-CO₂) is very effective in removing oils from a variety of seed matrices, devoid of any appreciable amount of phospholipid (PL) content. However, the limited solubility of PLs in SC-CO₂ leaves behind a potentially valuable by-product in the spent seed matrix. Any PL extraction process from the spent matrix must maintain the structure and the quality of PLs and must be compatible with the end use of the seed protein meal as animal feed or for human consumption. An initial SC-CO₂ extraction of soybean flakes was performed at 32 MPa and 80°C to extract the oil, leaving the PLs in the defatted soybean flakes (DSF). A second step was performed on the DSF using X_eth=0.10, varying the pressure from 16.6 to 68.9 MPa and the temperature from 60 to 80°C. For all SC-CO₂/ethanol extractions, a fraction rich in PLs was obtained. The fractions extracted from defatted soybean flakes were dried and then re-dissolved in chloroform before HPLC-ELSD analysis. Quantitative and qualitative analysis of PLs on soybean seeds, DSF, and different extracted phospholipid fractions was carried out, to ascertain the effort of combinations of extraction pressure and temperature.

Introduction

Phospholipids (PLs) are polar lipids 1, 2. The terms lecithin and phosphatidylcholine (PC) are often used interchangeably [2]. However, the term lecithin refers to a complex, naturally occurring mixture of PLs, traditionally obtained by water-washing crude vegetable oil and separating and drying the hydrated gums [3]. Therefore, the term lecithin is often used to describe a diverse group of commercially available PL mixtures, including fractions containing one or more PLs, triglycerides, pigments, carbohydrates, sterols, cerebrosides, in different proportions 3, 4.

Previous studies conducted at the National Center for Agricultural Research in Peoria, IL by Friedrich and co-workers 5, 6showed that supercritical carbon dioxide (SC-CO₂) was very effective in removing oils from a variety of seed matrices, devoid of any appreciable PL content. This property has recently been exploited by List et al. [7]to continuously degum pre-extracted soybean oil using SC-CO₂. However, the limited solubility of PLs in SC-CO₂ leaves behind a potentially valuable by-product in the spent seed matrix that could be recovered to economic advantage. In addition, any PL recovery process must also be compatible with the end use of the seed protein meal as an animal feed, or for use in human consumption. Since neat CO₂ will not effectively dissolve PL moieties, the choice of a suitable co-solvent to enhance their solubility must be made not only on a thermodynamic basis, but also with regard to its food safety status, i.e. `Generally Recognized As Safe' (GRAS). A logical choice for a co-solvent is ethanol, which enjoys GRAS status in the US. Ethanol has been used previously to fractionate PLs [8], although not in an SFE process; however, it has been utilized by Temelli [9]to remove the phospholipids from canola seed using SC-CO₂. Since high pressure phase equilibrium data are available for ethanol/CO₂ mixtures [10], we focused on the use of this co-solvent in fractionating the PL mixtures. The potential use of SC-CO₂/ethanol mixture for extraction and fractionation of PLs from defatted flaked soybean seeds has been investigated previously 11, 12. Initial studies indicated that a small amount of ethanol (5%) in the SC-CO₂ was not enough to extract the PLs, but by using a molar fraction of ethanol (X_eth) corresponding to 10%, the total recovery of PLs can be increased considerably. Otherwise, the relative amount of phosphatidylcholine (PC) and phosphatidyl-ethanolamine (PE) in the resulting extract can be varied, using amounts greater than 10%. Since SC-CO₂ extraction allows different kinds of products to be obtained by changing the operating parameters (pressure and temperature), the influence of these processing parameters on the extraction and fractionation was studied.

The individual properties of the constituents of lecithin are not well known, due to the difficulties in isolating large quantities of these compounds for study. For this reason it is highly desirable to obtain phospholipid-enriched fractions, and considerable effort is being put into developing schemes for phospholipid purification, particularly those based on the relative alcohol solubility of the individual phospholipids [13]. Moreover, the demand for high PC containing lecithin has been increasing in the cosmetic, pharmaceutical, food and other industries [14].

The objectives of this research were: (a) to evaluate the possibility of completely extracting the PLs present in the DSF by using a small amount of ethanol in SC-CO₂ (10% molar fraction), thereby confirming the possibility of carrying out a dual procedure for the removal of oil and phospholipid fractions from seed matrices using supercritical fluids; (b) to fractionate PLs by changing the pressure and the temperature of SC-CO₂/ethanol mixture. These objectives should be achievable considering that the solubility data for PC and PE in net ethanol (in subcritical status) show that PC is easier to dissolve than PE. Therefore, a first PC-enriched phospholipid fraction should be obtained; then, by changing the operating parameters, a second PC-depleted phospholipid fraction should be obtained.